Acanthamoeba myosin II rod is a long alpha-helical dimeric coiled-coil with a flexible hinge containing a helix-breaking proline. Previously, the thermal stability of the complete 'wild-type' rod domain of myosin II (residues 849-1509), a 'point' mutant in which the hinge proline was replaced with alanine (P398A), and a 'deletion' mutant with the whole hinge region deleted ( 384-408) in 0.6 and 2.2 M KCl, pH 7.5 was determined. In addition, analytical ultracentrifugation was used to characterize the size and shape (frictional ratio) of myosin II wild-type and mutant rods. In the present studies, sedimentation properties of minifilaments formed by dialysis of the wild-type and point and deletion mutants vs. low ionic strength buffer with 5 mM Mg (II) at pH 7.5 were determined. All three preparations of minifilaments were homogeneous with respect to size and shape. All three types of minifilaments were found to contain 8 coiled-coil rods per filament, in agreement with previous measurements showing that native myosin II forms antiparallel, octameric minifilaments. The calculated frictional ratios of the minifilaments of wild-type, point and deletion mutant rods corresponded to axial ratios for prolate ellipsoids of ca 83, 88, and 74, respectively. These values are somewhat larger than the value of 67 for the rod domain of native myosin II minifilaments estimated from published electron micrographs, suggesting the importance of volume and packing effects in filaments that were not included in frictional ratio calculations. It is noteworthy that calculated prolate ellipsoidal axial ratios of the monomeric coiled-coil point and deletion rods previously gave lengths of 90 nm, in excellent agreement with those measured by electron microscopy. The axial ratio of the wild-type myosin II rod was somewhat less, consistent with a flexible bend around Pro 398, which acts to effectively decrease the hydrodynamic frictional coefficient.